Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (A_L and B_L) or short (A_S and B_S) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AA_L and PDGF-BB_S isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.     

Temporal and functional changes in glycosaminoglycan expression during osteogenesis

Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.     

Relationships between glycosaminoglycan and receptor binding sites in chemokines-the CXCL12 example

Chemokines are small proteins, promoting directional migration and activation of different cells through binding to specific receptors. Most chemokines also bind to heparan sulfate (HS), a family of complex and highly sulfated glycosaminoglycan (GAG) found at the cell surface and in the extracellular matrix. This class of molecules has recently emerged as critical regulators of many events involving cell response to the external environment. Binding to HS is thought to be functionally important. Current models suggested that HS ensures the correct positioning of chemokines within tissues and maintains haptotactic gradients of the proteins along cell surfaces, thus providing directional cues for migrating cells. On the chemokine surface, the GAG binding epitopes can be displayed on different areas, some of which overlap the receptor binding domain, while others are clearly separated. We review here some structural aspects of the interaction between GAGs or receptors and chemokines. In particular, we will address the case of CXCL12, a chemokine whose receptor binding site is distinct from the GAG binding site and whose different isoforms display different GAG binding abilities. This chemokine system thus offers an unprecedented opportunity to ascertain the importance of chemokine/GAG interaction in the regulation of cell migration.     

Heparin binding by the HIV-1 tat protein transduction domain

The protein transduction domain from the HIV-1 tat protein (termed PTD-tat) has been fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G. We demonstrate that PG-Ctat (PTD-tat fused to the C-terminus of protein G) binds to a heparin affinity column. PG-Ctat binds with relatively high affinity, as shown by its elution at 1.6 M NaCl. The heparin binding properties of PTD-tat are consistent with the idea that heparan sulfate, an analog of heparin found at the cell surface, plays a role in the translocation of PTD-tat fusions. We suggest that the heparin-binding properties of PTD-tat can be exploited for purification of PTD-tat fusions in the absence of affinity tags.     

Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase/N-sulfotransferase 2

Heparin and heparan sulfate are linear sulfated polysaccharides that exert a multitude of biological functions. Heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase isoform 2 (NDST-2), a key enzyme in the biosynthesis of heparin, contains two distinct activities. This bifunctional enzyme removes the acetyl group from N-acetylated glucosamine (N-deacetylase activity) and transfers a sulfuryl group to the unsubstituted amino position (N-sulfotransferase activity). The N-sulfotransferase activity of NDST has been unambiguously localized to the C-terminal domain of NDST. Here, we report that the N-terminal domain of NDST-2 retains N-deacetylase activity. The N-terminal domain (A66-P604) of human NDST-2, designated as N-deacetylase (NDase), was cloned as a (His)(6)-fusion protein, and protein expression was carried out in Escherichia coli. Heparosan treated with NDase contains N-unsubstituted glucosamine and is highly susceptible to N-sulfation by N-sulfotransferase. Our results conclude that the N-terminal domain of NDST-2 contains functional N-deacetylase activity. This finding helps further elucidate the mechanism of action of heparan sulfate N-deacetylase/N-sulfotransferases and the biosynthesis of heparan sulfate in general.     

The effect of nitric oxide on metal release from metallothionein-3: gradual unfolding of the protein

Metallothionein-3 (MT-3), or neuronal growth inhibitory factor, which exhibits growth inhibitory activity, is a brain-specific metallothionein. In this study, the effect of nitric oxide (NO) on metal release (using Cd²⁺ as a probe) from MT-3 was examined by ¹¹³Cd and 2D [¹H–¹⁵N] heteronuclear single-quantum coherence NMR spectroscopy. The exposure of human MT-3 to NO leads to a nonselective release of the three metals from the β-domain. In contrast to metallothionein-1 and metallothionein-2, two of the bound metals in the α-domain were also partially released, with the domain structure remaining almost unchanged. Further addition of NO resulted in the complete release of metals and concomitant unfolding of the protein. The preference of release of the two metals in the α-domain was attributed to the presence of two slightly different coordination environments for the four cadmium/zinc atoms.     

Stage-dependent regulation of mammary ductal branching by heparan sulfate and HGF-cMet signaling

Specific interactions of growth factors with heparan sulfate may function as "switches" to regulate stages of branching morphogenesis in developing mammalian organs, such as breast, lung, salivary gland and kidney, but the evidence derives mostly from studies of explanted tissues or cell culture (Shah et al., 2004). We recently provided in vivo evidence that inactivation of Ndst1, the predominant N-deacetylase/N-sulfotransferase gene essential for the formation of mature heparan sulfate, results in a highly specific defect in murine lobuloalveolar development (Crawford et al., 2010). Here, we demonstrate a highly penetrant dramatic defect in primary branching by mammary epithelial-specific inactivation of Ext1, a subunit of the copolymerase complex that catalyzes the formation of the heparan sulfate chain. In contrast to Ext1 deletion, inactivation of Hs2st (which encodes an enzyme required for 2-O-sulfation of uronic acids in heparan sulfate) did not inhibit ductal formation but displayed markedly decreased secondary and ductal side-branches as well as fewer bifurcated terminal end buds. Targeted conditional deletion of c-Met, the receptor for HGF, in mammary epithelial cells showed similar defects in secondary and ductal side-branching, but did not result in any apparent defect in bifurcation of terminal end buds. Although there is published evidence indicating a role for 2-O sulfation in HGF binding, primary epithelial cells isolated from Hs2st conditional deletions were able to activate Erk in the presence of HGF and there appeared to be only a slight reduction in HGF-mediated c-Met phosphorylation in these cells compared to control. Thus, both c-Met and Hs2st play important, but partly independent, roles in secondary and ductal side-branching. When considered together with previous studies of Ndst1-deficient glands, the data presented here raise the possibility of partially-independent regulation by heparan sulfate-dependent pathways of primary ductal branching, terminal end bud bifurcation, secondary branching, ductal side-branching and lobuloalveolar formation.     

Current state on the enzymatic synthesis of glycosaminoglycans

Glycosaminoglycans (GAGs) are linear anionic polysaccharides, and most of them show a specific sulfation pattern. GAGs have been studied for decades, and still, new biological functions are discovered. Hyaluronic acid and heparin are sold for medical or cosmetic applications. With increased market and applications, the production of GAGs stays in the focus of research groups and the industry. Common industrial GAG production relies on the extraction of animal tissue. Contamination, high dispersity, and uncontrolled sulfation pattern are still obstacles to this process. Tailored production strategies for the chemoenzymatic synthesis have been developed to address these obstacles. In recent years, enzyme cascades, including uridine-5′-diphosphate sugar syntheses, were established to obtain defined polymer size and dispersity, as well as defined sulfation patterns. Nevertheless, the complex synthesis of GAGs is still a challenging research field.     

Selectivity in glycosaminoglycan binding dictates the distribution and diffusion of fibroblast growth factors in the pericellular matrix

The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in pericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the pericellular matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular matrix has long-range structure.     

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).     

Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [³⁵S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.     

Characterization of uniformly and atom-specifically C-labeled heparin and heparan sulfate polysaccharide precursors using C NMR spectroscopy and ESI mass spectrometry

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D ¹H NMR spectroscopy is often complicated by the extensive signal overlap. ¹³C NMR signals, on the other hand, overlap less and therefore, ¹³C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific ¹³C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly ¹³C- and ¹⁵N-labeled precursors were also produced and characterized by ¹³C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of ¹³C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% ¹³C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome-proteome interactions.     

Probing structural selectivity of synthetic heparin binding to Stabilin protein receptors

As one of the most widely used drugs worldwide, heparin is an essential anticoagulant required for surgery, dialysis, treatment of thrombosis, cancer, and general circulatory management. Stabilin-2 is a scavenger clearance receptor with high expression in the sinusoidal endothelium of liver. It is believed that Stabilin-2 is the primary receptor for the clearance of unfractionated and low molecular weight heparins in the liver. Here, we identify the modifications and length of the heparin polymer that are required for binding and endocytosis by both human Stabilin receptors: Stabilin-2 and its homolog Stabilin-1 (also found in liver endothelium). Using enzymatically synthesized (35)S-labeled heparan sulfate oligomers, we identified that sulfation of the 3-OH position of N-sulfated glucosamine (GlcNS) is the most beneficial modification for binding and endocytosis via both Stabilin receptors. In addition, our data suggest that a decasaccharide is the minimal size for binding to the Stabilin receptors. These findings define the physical parameters of the heparin structure required for efficient clearance from blood circulation. These results will also aid in the design of synthetic heparins with desired clearance rates.     

Radiolabeled heparan sulfate immobilized on microplate as substrate for the detection of heparanase activity

We developed a quantitative assay to monitor the enzymatic activity of heparanase, a protein responsible for the degradation of heparan sulfate (HS) present on cell surface and extracellular matrix. Our assay is based on a new procedure to immobilize radiolabeled HS to a solid support by a single end which is adaptable to a microplate format, thus allowing the rapid analysis of numerous samples. First, HS was radiolabeled by partial de-N-acetylation and re-N-acetylation with [3H] acetic anhydride, second, after reductive amination at the reducing terminus, it was covalently linked to an amino-reactive biotin analog, and third it was immobilized on a streptavidin-coated plate. The degradation of our solid-phase tritiated HS by heparanase was monitored by measuring the soluble radioactivity released in the well. The heparanase-induced release of radioactivity was linear with respect either to time or to the amount of enzyme and was inhibited by heparin or high ionic strength. The linearity of this assay for time and enzyme concentrations could be useful to determine the potency of heparanase inhibitors. Moreover, this assay was shown to be suitable for monitoring HS-degrading activity of either heparanase endogenously expressed by the HCT 116 tumor cell line or recombinant forms of this protein.     

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line ¹²⁵I-LDL, and on a murine macrophage cell line (J774) ¹²⁵I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.     

The cell surface of a restrictive fenestrated endothelium

Summary The location and chemical composition of anionic sites on the endothelium of the choriocapillaris was investigated with cationic ferritin and enzyme digestion techniques. Cationic ferritin administered intravenously initially labeled essentially all fenestral diaphragms. Within 30 min after injection, no diaphrams remained labeled, but they could be relabeled by a second cationic ferritin injection. Following perfusion of cationic ferritin, the entire luminal front of the endothelium was labeled: the plasmalemma and fenestral, vesicle, and channel diaphragms. Perfusion of neuraminidase or chondroitinase did not affect subsequent cationic ferritin binding. In contrast, heparitinase removed anionic sites on all structures except fenestral diaphragms. Cationic ferritin did not mark the endothelium following heparinase digestion. All sites were cleaved with pronase E. These results indicate that heparin is the anionic moiety on fenestral diaphragms while the glycocalcyces of the plasmalemma and vesicle and channel diaphragms are rich in a heparan sulfate proteoglycan. Furthermore, since the heparan sulfate localized to these structures was digested by both heparinase and heparitinase, it is in a form similar to heparin. These findings demonstrate that the endothelium of the choriocapillaris bears cell-surface anionic components that are different than those described for fenestrated endothelia lining other vascular beds.Supported by NIH EY 03776     

Effects of oxidative stress on the nuclear translocation of extracellular superoxide dismutase

The effect of oxidative stress on the cellular uptake and nuclear translocation of extracellular superoxide dismutase (EC-SOD) was investigated. EC-SOD was incorporated from conditioned medium of stable EC-SOD expressing CHO-EK cells into 3T3-L1 cells within 15 min. The uptake was clearly inhibited by the addition of heparin at a concentration of 0.4 microg/ml. Treatment of the 3T3-L1 cells with H(2)O(2) (5 mM for 5 min), followed by incubation with CHO-EK medium downregulated the uptake of EC-SOD. Nuclear translocation of the incorporated EC-SOD was clearly enhanced by H(2)O(2) treatment following incubation with the CHO-EK medium. EC-SOD is the only anti-oxidant enzyme which is known at this time to be actively transported into nuclei. The results obtained here suggest that the upregulation of the nuclear translocation of EC-SOD by oxidative stress might play a role in the mechanism by which the nucleus is protected against oxidative damage of genomic DNA.     

Interaction of chemokines and glycosaminoglycans: a new twist in the regulation of chemokine function with opportunities for therapeutic intervention

Despite their key role in inflammation, the apparent redundancy in the chemokine system is often cited as an argument against probing chemokines as therapeutic targets for inflammation. However, this in vitro redundancy frequently does not translate to the in vivo situation, as exemplified by the use of specific receptor antagonists, ligand neutralizing or receptor blocking antibodies and gene-deleted mice in models of human disease. Specificity may be conferred onto the chemokine system by fine-tuning of responses both temporally and spatially through their highly specific interactions with glycosaminoglycans (GAGs). In this survey, we present evidence for specificity in the interaction and introduce emerging technologies that enable detailed assessment of protein–GAG interactions. Finally, we address the issue of exploitation of this interaction for therapeutic advantage.     

Characterization of a synthetic peptide from the glycosaminoglycan binding site of heparin cofactor II

Summary We characterized a synthetic peptide based on the glycosaminoglycan (GAG)-binding site of the serine proteinase inhibitor (serpin) heparin cofactor II (HCII): HCII^165–195, K¹⁶⁵DFVNASSKYEITTIHNLFRKLTHRLFRRNF¹⁹⁵. HCII^165–195 negated acceleration of the HCII/thrombin inhibition reaction (IC₅₀ for the peptide shown in parentheses) by heparin (250 nM) and dermatan sulfate (500 nM). Circular dichroism spectra of HCII^165–195 showed that GAGs increase the -helical content of the peptide (percentage -helix of the peptide/GAG complex given in parentheses): no GAG (7%) < low-molecular-weight heparin (32%) < heparin (42%) < dermatan sulfate (55%). A molecular model of HCII predicts that this region is 48% -helix. Our results suggest: (i) HCII^165–195 binds to GAGs; (ii) an -helical conformation is preferable in the presence of GAGs; and (iii) GAGs may help stabilize a specific protein conformation in the HCII GAG-binding site, important for serpin function.     

The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling

Binding of growth factors to specific cell surface receptors is the first step in initiating cell signaling cascades that ultimately result in diverse activities such as proliferation, differentiation, and apoptosis. Dimerization and phosphorylation of tyrosine kinase transmembrane receptors is the typical paradigm for this activation but, for many growth factors, cell surface interactions are not limited to a single receptor type. In particular, heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG) on the cell surface and within the extracellular matrix (ECM), and these molecules have been viewed as accessory co-receptors serving to facilitate tyrosine kinase receptor binding. Recent studies, however, have indicated that HSPG can directly participate in signal transduction in response to FGF-2 binding. Thus, in the present study, we used mathematical modeling to examine whether the kinetics of formation of the various FGF-2 bound complexes on the cell surface correlate with the activation of the downstream mediators of FGF-2 response, Erk1/2. We find that FGF-2 binding to its receptor correlates well with Erk1/2 activation and that HSPG can modulate this response through its ability to stabilize these ligand receptor complexes. Moreover, we also observed that FGF-2 binding to HSPG correlates strongly with Erk1/2 activation under conditions where there is a loss of receptor activity, and we demonstrate that the relative amounts of signaling and non-signaling HSPG on the cell surface, as well as the presence of competing HSPG in the ECM, can impact the signal potential via this pathway. Thus, the selective regulation of specific HSPG might provide a mechanism for fine tuned modulation of heparin-binding growth factor signaling in cells where signal intensity and duration could direct cellular response toward growth, migration or differentiation.